Journal of Integrative Plant Biology 2008, 50 (11): 1406–1415
Effect of Elevated CO2 and Drought on Soil Microbial
Communities Associated with Andropogon gerardii
∗
Issmat I. Kassem1 , Puneet Joshi1 , Von Sigler1 , Scott Heckathorn1 and Qi Wang2
(1 Department of Environmental Sciences, University of Toledo, Ohio 43606, USA;
2
Department of Civil Engineering, University of Toledo, Ohio 43606, USA)
Abstract
Our understanding of the effects of elevated atmospheric CO 2 , singly and in combination with other environmental changes,
on plant-soil interactions is incomplete. Elevated CO 2 effects on C 4 plants, though smaller than on C 3 species, are mediated
mostly via decreased stomatal conductance and thus water loss. Therefore, we characterized the interactive effect of
elevated CO 2 and drought on soil microbial communities associated with a dominant C 4 prairie grass, Andropogon gerardii
Vitman. Elevated CO 2 and drought both affected resources available to the soil microbial community. For example, elevated
CO 2 increased the soil C:N ratio and water content during drought, whereas drought alone decreased both. Drought
significantly decreased soil microbial biomass. In contrast, elevated CO 2 increased biomass while ameliorating biomass
decreases that were induced under drought. Total and active direct bacterial counts and carbon substrate use (overall
use and number of used sources) increased significantly under elevated CO 2 . Denaturing gradient gel electrophoresis
analysis revealed that drought and elevated CO 2 , singly and combined, did not affect the soil bacteria community structure.
We conclude that elevated CO 2 alone increased bacterial abundance and microbial activity and carbon use, probably in
response to increased root exudation. Elevated CO 2 also limited drought-related impacts on microbial activity and biomass,
which likely resulted from decreased plant water use under elevated CO 2 . These are among the first results showing that
elevated CO 2 and drought work in opposition to modulate plant-associated soil-bacteria responses, which should then
influence soil resources and plant and ecosystem function.
Key words: denaturing gradient gel electrophoresis; drought; elevated CO 2 ; soil microbial communities.
Kassem II, Joshi P, Sigler V, Heckathorn S, Wang Q (2008). Effect of elevated CO 2 and drought on soil microbial communities associated with
Andropogon gerardii. J. Integr. Plant Biol. 50(11), 1406–1415.
Available online at www.jipb.net
During the last nine decades, the atmospheric concentration
of CO 2 has increased by 36%, primarily as a result of anthropogenic activities (IPCC 2007). Increased atmospheric CO 2
concentration and related environmental changes (e.g. warming
and decreased/increased precipitation) can influence the biological processes of plants and soil microorganisms inhabiting diverse ecosystems (Zak et al. 1993, 2000; Kandeler et al. 1998).
Elevated CO 2 typically increases plant growth in plants with both
Received 9 May 2008
Accepted 22 Jun. 2008
Supported by the University of Toledo Department of Environmental Sciences
and grants from the National Science Foundation to S. Heckathorn and E.W.
Hamilton.
∗
Author for correspondence.
Tel: +1 419 530 2897;
Fax: +1 419 530 4421;
E-mail: <[email protected]>.
C 2008 Institute of Botany, the Chinese Academy of Sciences
doi: 10.1111/j.1744-7909.2008.00752.x
C 3 and C 4 photosynthetic metabolism, though photosynthetic
and growth increases are generally larger in C 3 species (e.g.,
Sage and Monson 1999; Ehleringer et al. 2002; Wang et al. 2008
and references therein). In C 3 species, increased growth under
elevated CO 2 is primarily due to decreased photorespiration
(photosynthetic fixation of O 2 rather than CO 2 ), while in C 4
species, increased growth under elevated CO 2 is primarily due
to decreased stomatal conductance and transpiration, which
decreases soil water use (this also occurs in C 3 species).
As with most terrestrial ecosystems, grasslands will be impacted by future global environmental and climate changes,
including elevated CO 2 (Fay et al. 2003), and this will have
importance to the global ecosystem, as grasslands comprise
approximately one-quarter of the Earth’s land surface (Adam
et al. 2000). For example, under elevated CO 2 , Andropogon
gerardii, a dominant C 4 plant in tall-grass prairies, exhibits
increased above-ground biomass (19–166% under favorable
conditions), photosynthesis, and root exudation (in mixed plant
communities), as well as decreased stomatal conductance (in
wet seasons), sap flow and evapotranspiration (Owensby et al.
Effect of CO 2 and Drought on Microbes
1994, 1997, 1999; Adam et al. 2000; Williams et al. 2000,
2004). Grassland soils are naturally water-limited (Knapp 1984).
Therefore, small increases in soil water content brought about
by elevated CO 2 (Adam et al. 2000) can have a dramatic
impact on microbial biomass production and nutrient use (Rice
et al. 1994; Griffiths et al. 2003). It follows that elevated CO 2
and its subsequent impact on A. gerardii physiology might
significantly alter critical microbial functions in grassland soils,
including organic matter decomposition, carbon release, and the
availability of plant nutrients (Zak et al. 2000; Alkorta et al. 2003;
Griffiths et al. 2003; Arias et al. 2005). Despite this relationship,
few studies have addressed the indirect effect of elevated CO 2
on both structure and activity of soil microbial communities
associated with plants (Ebersberger et al. 2004).
Increased atmospheric CO 2 concentration affects the occurrence of droughts (Ward et al. 1999), which have increased
in duration and intensity worldwide since the 1970s (IPCC
2007). Drought impacts ecosystem functions and plant-microbe
relationships in terrestrial environments (Griffiths et al. 2003;
IPCC 2007), it also affects the partitioning of carbon between
the atmosphere and terrestrial systems. For example, prolonged
drought in North America in 2002 reduced the amount of carbon
uptake by vegetation and soil from an annual average of 650
million metric tons to 330 million metric tons (Peters et al. 2007).
Since the frequency of heat and drought stresses is predicted to
increase (Mearns et al. 1984), it follows that the amount of carbon sequestered by terrestrial ecosystems will likely decrease.
Consequently, the combination of increased drought and CO 2
enrichment could result in a chronic impact on soil microbial
communities and their relationships with plants. However, to
our knowledge, the effect of this important interaction on the
structure and activity of soil microbial communities has not been
examined.
Because of the complexity of plant-soil interactions, a multiphasic approach is necessary to understand the relationship between elevated CO 2 , singly and in combination with
drought, and plant/soil microbial communities. Many previous
studies used traditional microbiological techniques to identify
changes in soil bacteria in response to elevated CO 2 concentration. The results of these studies were conflicting, as no
response, increases, and decreases in biomass were reported
(Diaz et al. 1993; Zak et al. 1993, 2000; Allen et al. 2000;
Williams et al. 2000; Freeman et al. 2004). These inconsistent
conclusions are likely attributable to natural variation in soil
microbial communities, soil type, the species of plant under
study and methodological biases (Sadowsky and Schortemeyer
1997; Franklin and Mills 2003; Schmidt 2006). Although no
techniques are without bias (Muyzer and Smalla 1998), the
combination of molecular fingerprinting with traditional methods
can yield a more comprehensive characterization of microbial communities, as compared with traditional methods alone
(Torsvik and Ovreas 2002; Schmidt 2006). A popular method
for characterizing impacts to complex microbial assemblages
1407
is denaturing gradient gel electrophoresis (DGGE) analysis
(reviewed in Muyzer and Smalla 1998), which has been used
previously to assess the impact of elevated CO 2 on the structure
of soil microbial communities. For example, studies conducted
by Ebersberger et al. (2004) revealed that CO 2 enrichment
resulted in significant season-specific changes in community
structure, suggesting that the effect of elevated CO 2 on soil
microbial communities might be modulated by the presence of
other prevailing environmental parameters.
To better understand the potential effects that global climate
change might have on soil microbial communities, particularly
in C 4 -dominated grasslands, we investigated the interactive
effect of elevated CO 2 and drought on the activity and structure
of the soil microbial community associated with A. gerardii, a
dominant, drought-resistant grass of central US prairies (Knapp
1985). We hypothesized that elevated CO 2 would increase
soil water availability, thereby mitigating the effect of drought
on the microbial communities. To test this hypothesis, we
used a multiphasic approach that incorporated molecular and
traditional microbiology to assess the plant-mediated impact of
elevated CO 2 and drought in the context of bacteria abundance,
activity and community structure.
Results
Plant physiological responses
Leaf water potential, a measure of plant water status, decreased
substantially with drought (P < 0.001), and there was significant
drought x CO 2 interaction (P < 0.001), such that elevated CO 2
largely ameliorated the decrease in water potential with drought
(Table 1); similar results were observed for leaf relative water
content (not shown). Shoot mass was decreased (P = 0.09) and
root mass increased by drought (P = 0.009), while both were
unaffected by CO 2 ; thus, the gradual drought treatment stimulated root growth in this drought-tolerant plant species. Stomatal
conductance was decreased by both drought (P = 0.08) and
elevated CO 2 (P = 0.001), and there was an interactive effect
(P < 0.05), such that stomatal conductance was lowest in the
combined elevated CO 2 and drought treatment. Net photosynthesis was decreased by drought (P < 0.016) and increased by
elevated CO 2 (P < 0.001), and there was a significant drought
x CO 2 interaction (P < 0.004), such that photosynthesis did not
decrease during drought under elevated CO 2 .
Plant and soil C:N
The C:N ratio of plant shoots was not significantly affected by
either drought or CO 2 (Figure 1); similar results were observed
for both shoot %N and %C (not shown). In contrast, root C:N
was decreased by drought (P = 0.007) and was unaffected by
elevated CO 2 ; this decrease in C:N was caused by a decrease
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Vol. 50 No. 11
2008
Table 1. Summary of plant parameters across treatments
Stomatal conductance
Photosynthesis
Water potential (MPa)
Shoot biomass (g)
Root biomass (g)
(mol H 2 O/m2 per s)
(μmol CO 2 /m2 per s)
Control
−1.34 ± 0.10
2.95 ± 0.38
2.75 ± 0.39
0.16 ± 0.02
22.35 ± 0.96
Drought
−3.55 ± 0.44
2.43 ± 0.13
7.26 ± 1.99
0.13 ± 0.01
16.97 ± 0.45
Elevated CO 2
−1.51 ± 0.13
2.84 ± 0.31
2.82 ± 0.35
0.09 ± 0.01
27.2 ± 0.30
Elevated CO 2 and drought
−1.74 ± 0.07
2.19 ± 0.22
4.66 ± 0.50
0.05 ± 0.01
29.97 ± 0.81
Treatment
The results are reported as mean values ± SE across at least four replicates.
Active bacteria. The number of active bacteria present
under control conditions was statistically similar to the number
observed under drought conditions (P = 0.34) (Figure 2A). The
number of active bacteria present under the elevated CO 2 treatment was significantly higher (P < 0.05) than the number under
control and drought treatments, but not significantly different
(P = 0.32) than the number observed following treatment with
combined elevated CO 2 and drought. Additionally, the number
of active bacteria observed in the combined elevated CO 2 and
drought treatment was significantly higher than the number
observed in control or drought treatments (P < 0.05).
50
45
40
C:N ratio
35
30
25
20
15
10
5
0
Control
Drought
Elevated
CO2
Drought +
elevated CO2
Figure 1. C:N ratio measured for shoots ( ), roots () and bulk soil ()
under drought and elevated CO 2 .
The results are reported as mean values ± standard error (n = 3).
in %C with drought (P = 0.047), as %N was unaffected by
the treatments (not shown). The C:N ratio of bulk soil was
increased by elevated CO 2 (P = 0.005) and decreased by
drought (P = 0.008) (Figure 1). These changes were due to
small, but statistically insignificant, increases in %C resulting
from elevated CO 2 and decreases with drought (ambient and
high CO 2 ), and decreases in %N with elevated CO 2 (not shown).
For rhizosphere soil (adhering to roots), C:N increased with CO 2
and was unaffected by drought (not shown).
Estimation of total and active bacteria abundance
Total bacteria. The number of soil bacteria (cells/g dry soil)
under control and drought treatments were statistically similar
(P = 0.48) (Figure 2A). Likewise, the number of soil bacteria
observed under the elevated CO 2 treatment was similar to that
resulting from combined elevated CO 2 and drought (P = 0.32).
However, the number of bacteria observed under conditions of
elevated CO 2 (including combined elevated CO 2 and drought)
were significantly higher than the number observed under other
treatments including ambient CO 2 concentrations (P < 0.000 1).
Estimation of microbial biomass by quantifying soil DNA
The concentration of DNA isolated from the soils (μg DNA/g dry
soil) was used to estimate soil microbial biomass (Garcia-Pichel
et al. 2003). Specifically, the concentration of DNA isolated
from control soils was significantly higher than that isolated from
soils under drought (P < 0.000 1) and combined elevated CO 2
and drought (P < 0.01) conditions. Although the concentration
of DNA isolated from soils under elevated CO 2 was similar
to that observed under control conditions (P = 0.17), it was
significantly higher than in soils under drought (P < 0.000 1)
and combined elevated CO 2 and drought (P < 0.01) treatments.
Finally, the concentration of DNA isolated from soils under
drought conditions was significantly lower than from soils under
combined elevated CO 2 and drought (P < 0.05).
Soil microbial activity
The rate of FDA hydrolysis (μg fluorescein produced per g
dry soil per h) was used to estimate the overall enzymatic
activity of the soil microbial community (Adam and Duncan
2001). Overall, microbial FDA hydrolysis activity in soils under
the drought treatment was higher, as compared with all other
treatments (all comparisons P < 0.05), while combined elevated
CO 2 and drought resulted in the lowest activity (Figure 2C).
Microbial activity under control conditions was similar to that
under elevated CO 2 (P = 0.33), and combined elevated CO 2
and drought (P = 0.056) treatments. Microbial activity was
significantly higher under the elevated CO 2 treatment than
under combined elevated CO 2 and drought (P < 0.05).
6
A
d
d
5
4
b
b
3
2
1
c
a
a
c
0
700
B
600
a
a
500
c
b
400
300
200
100
0
Average fluorescein produced
(µg/g dry soil per h)
1409
of all other treatments (P < 0.05), while the lowest CMD was
measured under the drought and control treatments (Figure 3A).
In addition, the CMD measured under combined elevated CO 2
and drought was higher than that measured under control
conditions (P < 0.05), but was not significantly different than
that measured under drought (P = 0.15) conditions. The CMD
measured under the drought treatments was similar to that
measured under control conditions (P = 0.32).
The average metabolic response (AMR) exhibited a similar
trend to CMD, as the highest AMR was measured under the
elevated CO 2 treatment (Figure 3B). Specifically, the AMR under elevated CO 2 was significantly higher than the AMR under
the control, drought, and combined elevated CO 2 and drought
treatments (all P < 0.05). The AMR resulting from combined
elevated CO 2 and drought was similar to that measured under
drought (P = 0.22) and control (P = 0.299) conditions. Finally,
no significant difference in AMR was detected between control
and drought treatments (P = 0.25).
120
C
b
110
80
60
a,c
c
a
40
Average number of used substrates
produced (µg/g dry soil per h)
Average number of active and total
cells (108) per g dry soil
Effect of CO 2 and Drought on Microbes
35
A
c
30
b
a,b
25
a
20
15
10
5
0
20
1.4
B
0
Drought
Elevated Drought +
CO2 elevated CO2
Figure 2. The effect of drought and CO 2 exposure on the enumeration
and activity of the soil microbial biomass.
(A) Number of total ( ) and active () bacteria.
b
1.2
Optical density
Control
1
0.8
a
a
Control
Drought
a
0.6
0.4
(B) Microbial biomass (concentration of soil DNA).
(C) Microbial community activity (FDA analysis).
Same letter designation indicates that no statistical significance was
observed (α = 0.05). The results are reported as mean values ± SE
0.2
0
(n = 3).
Bacteria metabolic diversity and average
metabolic response
The metabolic diversity of culturable bacteria (CMD) following
the elevated CO 2 treatment was significantly higher than that
Elevated
Drought +
CO2
elevated CO2
Figure 3. Response of microbial activity to drought and elevated CO 2
as assessed by (A) bacteria community metabolic diversity, and (B)
average metabolic response.
The results are reported as mean responses ± standard error (n = 3).
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Vol. 50 No. 11
2008
Figure 4. Unweighted pair-group with arithmetic means algorithm (UPGMA) dendrogram showing the percent similarity of soil bacteria communities
subjected to CO 2 and drought treatments.
Bacteria community structure
Denaturing gradient gel electrophoresis analysis of soil bacteria
resulted in fingerprints that exhibited an average of 48 dominant
bands, which did not change significantly (P < 0.05) across
treatments. All profiles were 100% similar (Dice coincidence
index), exhibiting no treatment-based differences (Figure 4).
Discussion
The overall objective of this study was to determine the impact of
elevated atmospheric CO 2 , with and without drought stress, on
soil microbial communities, especially bacteria, associated with
A. gerardii. Our data provide evidence that soil water availability,
soil carbon and nitrogen levels, or C:N ratio might be primary
factors influencing the activity and abundance of soil bacteria
associated with A. gerardii. Specifically, elevated CO 2 appeared
to decrease/mitigate the negative impacts of drought on microbial communities by decreasing the stomatal conductance
of A. gerardii (Table 1), which subsequently increased water
availability to soil microorganisms. This corroborates previous
findings that showed decreases in stomatal conductance in
A. gerardii (Owensby et al. 1997; Adam et al. 2000; Williams
et al. 2000), increases in soil water content (Wullschleger et al.
2002; Lecain et al. 2003) and soil C:N ratio under elevated CO 2
(Williams et al. 2000; Phillips et al. 2006), which collectively
were predicted to result in increased microbial growth (Zak et al.
1993, 2000). Our findings show that the increased soil C:N ratio
(Figure 1) was driven by a small increase in %C under elevated
CO 2 and a small decrease in %N under drought. Since elevated
CO 2 increased photosynthesis, but did not increase plant C:N
or mass, the increase in soil C:N ratio and %C probably resulted
from increased root carbon exudation.
Griffiths et al. (2003) and Whiteley et al. (2003) showed
that water stress decreased the number of culturable bacteria
in grassland soils. However, our data showed that moderate
drought did not appear to impact the number of total and active
bacteria (Figure 2A). Since direct counts of “active” cells have
been shown to actually provide an indication of cell viability
rather than activity (Creach et al. 2003), it is likely that drought
decreased the culturability of the soil bacteria, which would support the results reported by Griffiths et al. (2003) and Whiteley
et al. (2003). As shown by DNA quantification, drought appeared
to decrease soil microbial biomass, whereas the addition of
elevated CO 2 exhibited a mitigating effect, significantly increasing microbial biomass and the abundance of active and total
bacteria (Figure 2A,B). This response can be at least partially
explained by noting that water stress can result in bacteria
lysis (Fierer et al. 2003), which subsequently releases nutrients
that feed surviving microorganisms and increase their activity
(Figure 2C). An alternative explanation is that drought induced
Effect of CO 2 and Drought on Microbes
an increase in the concentration and diversity of intercellular
enzymes capable of FDA hydrolysis in the soil matrix (Halverson
et al. 2000). Although the FDA assay provides an appropriate
measure of overall microbial activity, it should be noted that this
assay is not bacteria-specific (Breeuwer et al. 1995). Therefore,
other soil organisms such as lichens and fungi could have
contributed to the observed increase in overall activity (Palmer
and Friedmann 1990; Gaspar et al. 2001). Regardless of the
cause, increased activity following drought appears to be aided
by the tolerance of the grassland microbial communities to
conditions of water limitation (Knapp 1984; Griffiths et al. 2003).
It was notable that communities exposed to combined elevated
CO 2 and drought exhibited significantly lower FDA activity than
those under drought stress alone. This result also suggested
that elevated CO 2 played a role in decreasing/mitigating the
effect of drought, likely by limiting evapotranspiration and cell
lysis or by increasing root exudation by A. gerardii.
It is established that bacteria in grassland soils are tolerant to
water limitation (Fierer et al. 2003; Griffiths et al. 2003). Therefore, it was not surprising that following the drought treatment,
the degree and diversity of substrate use by culturable soil
bacteria was unchanged relative to the control (Figure 3A,B).
In contrast, these parameters significantly increased under
elevated CO 2 , which could result from CO 2 -driven increases
in root carbon exudation and soil water content (Diaz et al.
1993; Dhillion et al. 1996). The combination of elevated CO 2
and drought appeared to moderate the degree and diversity of
substrate use, likely due to several interacting factors including a
balance between changes in root biomass, soil water availability
and soil C:N. Collectively, these results show that multiple
environmental factors can work in opposition as well as in
concert to modulate the activity of bacteria. Our metabolic
analysis was intended not as an absolute measure of in situ
microbial activity, but as an index by which to compare treatment
impacts. It should be noted that this analysis does not select
for the soil microbial community as a whole, but for a subset
of the community that will exhibit growth on a defined substrate
(Konopka et al. 1998). Therefore, while our results do not reflect
the contribution of all bacteria, they can provide a valuable
index of relative functional potential and an effective means
to compare the culturable subsets of differing communities of
bacteria (Haack et al. 1995).
Previous studies have indicated that grassland microbial communities were resistant to structural changes following exposure
to either elevated CO 2 (Bruce et al. 2000; Ebersberger et al.
2004) or water stress (Griffiths et al. 2003). While corroborating
those findings, our results also expand on them by providing evidence that the interactive effect of elevated CO 2 and
drought on the bacteria community structure was also limited.
Despite the likely stimulatory effect of CO 2 enrichment on root
exudation and its subsequent impact on the activity of some
soil bacteria, exudation might not provide enough carbon to
drive gross structural changes in the bacteria community (Bruce
1411
et al. 2000). Additionally, Griffiths et al. (2003) predicted that
although sudden changes in soil moisture potential might result
in bacteria death, gradual soil drying could result in adaptation to
water stress. This phenomenon would explain the limited impact
to the bacteria community structure of the drought treatment,
which was designed to mimic the slow onset of natural drought.
The addition of elevated CO 2 might further offset the impact of
drought by effectively increasing water availability and therefore
allowing soil bacteria a further opportunity to adapt to water
stress. While DGGE analysis is an effective method for detecting
substantial changes in diverse microbial assemblages (Muyzer
et al. 1993), it should be noted that subtle changes to complex
communities might go undetected (Bruce et al. 2000; McCaig
et al. 2001; Ebersberger et al. 2004). Therefore, we maintain
that drought and elevated CO 2 , singly and in combination, had
a limited effect on the gross community structure of bacteria
associated with A. gerardii.
While previous studies have more or less addressed the
individual impact of drought and elevated CO 2 on microbial communities (Bruce et al. 2000; Griffiths et al. 2003; Ebersberger
et al. 2004), their interactive effects have not been investigated.
Our results provide evidence that elevated CO 2 can mitigate
the negative impacts of drought on the biomass and activity of
soil microbes as well as certain plant properties. Furthermore,
this study revealed that elevated CO 2 and drought, singly and in
combination, had a limited impact on the microbial community
structure. As atmospheric CO 2 concentrations increase along
with the frequency of droughts (Mearns et al. 1984), a delicate
interplay between detrimental and mitigating effects will impact
the response of microbial communities. Therefore, the impact
of increased CO 2 and drought on ecosystem functions should
not be examined as independent phenomena, but in concert, as
well as with other drivers of environmental changes.
Materials and Methods
Experimental design
Andropogon gerardii Vitman (big bluestem) was germinated
from seeds collected at the Konza Prairie Research Natural Area (Manhattan, KS, USA). Following germination, the
seedlings were transferred to pots (53 cm deep × 10 cm
diameter) containing soil collected from mesic prairies in the Oak
Opening Metropark (Toledo, OH, USA) that were dominated by
A. gerardii. Prior to use, the soil was homogenized by thorough
hand mixing while wearing sterile gloves and sieved (2 mm
opening size). The plants were arranged into four groups each
containing five replicate pots. The plants were subjected to one
of four treatments for 70 d: (i) control (incubation at 370 μmol/mol
CO 2 , ambient concentration); (ii) elevated CO 2 (incubation at
700 μmol/mol CO 2 , approximately double the ambient concentration); (iii) moderate drought (Heckathorn et al. 1997)
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Vol. 50 No. 11
(following one month of daily watering, no water was provided
for 40 d); and (iv) both elevated CO 2 treatment and drought
combined. The elevated CO 2 treatments were conducted in
environment chambers (E-36HO, Percival Scientific, Perry, IA,
USA) equipped to maintain desired light and temperature levels
and CO 2 concentration. No fertilizer was added throughout the
experiment and all groups were treated with the same temperature regime (30 ◦ C day and 22 ◦ C night). Light levels were
maintained at 700–1 000 μmol/m2 per s PPFD (photosynthetic
photon flux density) during the experiment.
Measurement of plant parameters
The stomatal conductance to water vapor and net photosynthesis (i.e. CO 2 uptake) was measured using a portable infrared
gas analyzer (Li-Cor 6400, Lincoln, NE, USA) according to the
method of Heckathorn et al. (1996). Leaf water potential was
measured using a pressure chamber (Heckathorn et al. 1997)
before harvesting the plants for further analyses. Relative water
content was measured after harvesting the plants, and was
calculated from the fresh, dry, and turgid weight of plant leaves.
Turgid weight was determined after soaking the leaves in
distilled water overnight at low-light conditions, while dry weight
was measured after incubating the leaves at 70 ◦ C for 72 h.
Dried roots and shoots were ground separately using a mortar
and pestle, and sieved (0.5 mm mesh-pore) for C:N elemental
analysis using a Perkin-Elmer 2400 CHNS/O Analyzer (Series
II), following the manufacturer’s specifications. All analyses
were carried out in triplicate at the end of the incubation period.
Soil processing and storage
After plant collection, the bulk and rhizosphere soils from each
pot were sieved (2 mm) and thoroughly homogenized by hand
mixing (using sterile gloves). Soil samples were assessed in
duplicate for C:N elemental analysis as described above. Soils
used for bacteria counts and enzymatic activity assays were
stored at 4 ◦ C and processed within 12 h of collection, while soil
samples for DNA-related experiments were immediately frozen
at −70 ◦ C for subsequent analyses.
Estimation of the abundance of total and active bacteria
Three grams of each soil sample were added to 150 mL of
10 mmol/L sodium phosphate buffer (pH 7.0) and shaken for
1 h. CTC (5-cyano-2, 3-di-4-tolyl-tetrazolium chloride) (Polysciences, Inc., PA, USA) (200 μL,) was added to 800 μL of
soil slurry (5 mmol/L final concentration) and incubated for 16
h in the dark (Gruden et al. 2003). Cells were fixed by adding
formaldehyde (2% final concentration) and incubating for 5 min
(Kepener et al. 1994) at room temperature, followed by the
addition of Tween 80 (0.1% final concentration). Samples were
incubated for 5 min at room temperature and then sonicated for
2008
4 min (45 s pulses at 160 W) (Griebler et al. 2001), to separate
bacteria from soil particles. The samples were centrifuged at
50g for 12 min and 800 μL of supernatant was transferred into
a new tube, and then stained with 0.5% Picogreen (Quant-iT
dsDNA reagent, Molecular Probes) for 5 min (Kepener et al.
1994; Griebler et al. 2001). The supernatant was filtered onto
GE polycarbonate (PCTE) filter membranes (pore size 0.2 μm)
(Washington DC, USA) under low vacuum pressure. The filters
were transferred onto microscope slides for examination. Cells
were visualized with fluorescence microscopy (1 000× magnification) to enumerate total cells (those stained with Picogreen)
and active cells (cells that reacted with CTC). Ten random fields
were selected or an equivalent of a minimum of 200 bacterial
cells was counted for each slide. The results were reported as
total- and active bacteria per gram dry soil.
Fluorescein diacetate hydrolysis assay
Overall microbial activity was measured by determining the rate
of fluorescein diacetate (FDA) hydrolysis. Three grams (wet
weight) of soil were suspended in 15 mL of 60 mmol/L sodium
phosphate buffer (pH 7.6). FDA (150 μL of a 1 000 μg/mL
solution, dissolved in acetone) was added to each sample.
Two controls were also used, including one that contained soil
and buffer only (to account for background fluorescence in the
absence of FDA), and another prepared without soil, containing
only buffer and FDA (to determine the rate of background
fluorescein production in the absence of soil). All samples were
incubated in a shaking water bath (30 min at 200 r/min) at
30 ◦ C, following which enzymatic activity was stopped by briefly
shaking with 15 mL of 2:1 chloroform/methanol. Each sample
was then subjected to centrifugation for 5 min at 3 000g followed
by filtration of the supernatant through filter paper (Whatman,
No. 3). The resulting solution was measured for absorbance at
OD 490 .
The absorbance of each sample was compared with those of
fluorescein standard solutions prepared as described by Adam
and Duncan (2001). All samples and controls were assayed in
triplicate and the results were expressed as μg of fluorescein
produced per gram dry soil per hour.
Bacteria metabolic diversity and average
metabolic response
One hundred microliters of each soil (diluted to contain approximately 104 bacteria) sample were added to each well of
a BIOLOG Ecoplate (Garland and Mills 1991). Plates were
incubated in a humidified chamber for 48 h at 25 ◦ C, after
which the optical density (OD 590 ) was determined using a
Model 680 microplate reader (Bio-Rad, CA, USA). Raw and
background absorbance data were collected and analyzed
using Microsoft Excel to measure two parameters. First, the
AMR, which provides an index of the overall carbon substrate
Effect of CO 2 and Drought on Microbes
use by culturable bacteria, was determined by calculating the
average OD 590 per plate. Second, CMD was used to estimate
the metabolic diversity of culturable bacteria. CMD refers to the
number of differing carbon sources used by the bacteria and
was calculated by summing the number of wells in which the
absorbance exceeded an OD 590 of 0.2 (Lohner et al. 2007).
1413
commonly used in our laboratory. The DGGE markers were
loaded such that a maximum of two samples separated each
marker lane. Similarities among fingerprints were identified by
constructing a matrix of the Dice coincidence index (Dice 1945).
A dendrogram describing the similarity matrix was created using
cluster analysis, which was carried out using the unweighted
pair-group with arithmetic means algorithm (UPGMA).
DNA isolation from soil
Statistical analysis
Approximately 0.8 g (wet weight) of each soil, 0.5 mL of 0.1 mm
glass beads (Biospec Products Inc., OK, USA) and 1 mL of
sterile DNA extraction buffer (pH 8) (50 mmol/L NaCl, 50 mmol/L
Tris-HCl (pH 7.6), 5% sodium dodecyl sulfate (SDS)) were
added to a 2 mL microcentrifuge tube and shaken in a bead
beater (Fastprep, Bio101, Thermosavant, MN, USA) for 30 s at
5.5 m/s (Sigler and Zeyer 2004). DNA isolation was carried
out using phenol (pH 8)/chloroform extraction (as described
in Sambrook and David 2001). Further DNA purification was
carried out by passing the DNA solution through a column
containing PVPP (polyvinylpyrrolidone, Acros Organics, NJ,
USA) (Kuske et al. 1998). Isolated DNA was quantified by
measuring optical density at A 260 and diluted with DNA-grade
water to a concentration of 50 μg/mL before further use.
Differences in means were assessed with two-way ANOVA followed by Tukey’s test to identify significant differences among
treatments. Significance was determined at α = 0.05.
Acknowledgements
We thank Malak A. Esseili for her critical review of the
manuscript. We acknowledge the support of the Lucas County
Metroparks, who provided access to soil sampling sites.
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